Adenovirus E1A protein is directly involved in oncogenic transformation by adenovirus. It is a nuclear protein with structural and functional homology to the product of the important myc human oncogene. E1A protein stimulates transcription of genes introduced into mammalian cells by transfection or viral infection. The mechanism of this process, called E1A transactivation, is not yet well understood. Results from the first funding period indicate that specific host cell transcription factors including TFIIIC1, TFIIIC2 (both identified during the first funding period), and the TATA-box transcription factor increase in activity after expression of E1A. However, none of these host cell transcription factors have yet been purified to homogeneity. Consequently, it has not been possible to directly determine the effect of E1A protein on the quantity and specific activities of these transcription factors. To do this, and to pursue the biochemical mechanisms by which the activities of these transcription factors are increase by E1A protein, we propose to purify these transcription factors to homogeneity. This will enable a direct characterization of the polypeptides which comprise these activities. Specific antisera will be raised against the purified proteins and used to determine the amounts and physical properties of the proteins in adenovirus and mock infected HeLa cells. Purified transcription factors will also be used in studies of the functions of the transcription factors in transcription factors in transcription initiation. Interactions of TFIIIC2 and TFIIIC1 with wild-type and mutant VAI, tRNA, and 5S rRNA genes will be analyzed by nuclease protection and binding interference studies. The interaction of the TATA-box factor with wild-type and mutant Ad2 E1B promoters which have been well characterized for their transcription in vivo will be analyzed. cDNAs and genes encoding the TFIIIC1, TFIIIC2 and TATA-box TF polypeptides will be isolated and analyzed. This will yield information on the amino acid sequence of the proteins which will be useful in their characterization in infected and uninfected cells. For example, it is possible that these proteins are regulated by post- translational modifications such as phosphorylation. Amino acid sequence would be crucial to defining the sites of modification. Genomic and cDNA clones will also be useful in analyzing the transcriptional regulation of these genes in response to E1A and growth factors. Results form the proposed studies should contribute to an understanding of transcription factor function and regulation and the mechanism of E1A transactivation.